Reference may be made to WHO, 1989, WHO expert committee on Rabies: WHO Technical Report, WHO, Geneva reporting that rabies is one of the most important and widespread zoonotic diseases and is, with the exception of a few countries, a truly global problem. Rabies is a dreadful disease, one of the oldest known to mankind. It spreads due to the bite of an infected animal or by mucosal exposure. Once the symptoms appear, it proves fatal.
Reference may be made to Hohnes et al., 2002; “Genetic Constraints and the Adaptive Evolution of Rabies Virus in Nature” Virology 292, 247-257 who used a molecular evolutionary approach to investigate the adaptation of rabies virus in nature. Their analysis revealed that the DNA sequence of the nucleoprotein (N) and glycoprotein (G) genes of natural viral isolates were highly constrained, especially at nonsynonymous sites, in contrast to the higher rates of nonsynonymous evolution observed in viruses subject to laboratory passage. In evidence to this statement Charlton et al., 1997. “The long incubation period in rabies: Delayed progression of infection in muscle at the site of exposure”, Acta Neuropathologica. 94, 73-77 reported that although rabies virus has a strong neurotropism, replication in vivo does not take place only in neuronal cells. In particular, the virus replicates in muscle tissue also at the site of inoculation before entering the peripheral and central nervous system as well as the salivary gland and other non nervous tissues. Such a process has been documented by Morimoto et al., 1996. “Characterization of a unique variant of bat rabies virus responsible for newly emerging human cases in North America” Proc. Natl. Acad. Sci. USA 93, 5653-5658 who state that in vitro substitutions in the viral glycoprotein (G) sequence accumulate in cell culture of rabies virus and change the tropism for nervous tissues, thereby changing virulence. This shows an adaptive process of virus which was also highlighted in study of Kissi et al., 1999 “Dynamics of rabies virus quasispecies during serial passages in heterologous hosts”, J. Gen. Virol. 80, 2041-2050 who observed substantial genetic variation in the G protein coding gene from viruses passaged through different host species.
Reference may be made to Patrick et al., 1987. U.S. Pat. No. 4,707,356 wherein, a peptide vaccine requires identifying an antigenic determinant on the virus which has a sequence that is highly conserved among the various strains. A segment of the rabies virus coat glycoprotein was discovered which has a sequence homologous with the conserved sequence of the segment of the curaremimetic, snake—venom neurotoxins which includes the segment through which the toxins are thought to bind to the acetylcholine receptor binding—site (the AchR) at neuromuscular junctions. Lentz et al. reported in Science 226, 847-848 (1984) “Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins” that rabies virus accumulates at the neuromuscular junction by binding to the acetylcholine receptors at such junction. Findings reported by Lenz et al. are that the binding of rabies virus at the neuromuscular junction can be blocked by pre-incubation of tissue including such junctions with the curaremimetic, snake—venom neurotoxin, alpha—bungarotoxin, which is known to bind tightly to the acetylcholine (Ach) binding site of the AchR.
Reference may be made to Dietzschold et al., 1979. “Rabies virus strain. A comparative study by polypeptide analysis of vaccine strain with different pathogenic patterns” Virology 98 63-75 wherein five constituent polypeptides of the rabies vaccine strains ERA, HEP, CVS and PM (Stereotype I) and Mokola (Serotype 3) were examined by tryptic peptide analysis and revealed general similarity between the nucleoproteins of Mokola and four serotype 1 strains while overall comparison of the tryptic vaccine strains indicates that CVS and PM are more closely related to each other than to ERA or HEP.
Reference may be made to Slater Aug. 9, 1977 U.S. Pat. No. 4,040,904 “Novel rabies virus vaccine and processes” wherein, the ERA strain of the rabies virus was derived from SAD virus strain, originally isolated from a rabid dog and propagated in mouse brain and hamster kidney cells and then adapted to primary pig kidney tissue culture. A sample of the ERA strain of rabies virus was deposited with the American Type Culture Collection, Washington, D.C. on Oct. 29, 1964 and was recorded there as number VR 332. Vaccines containing the ERA strain adapted to primary pig kidney tissue culture are widely used in immunizing various animal species including dogs, cats and cattle against rabies. According to the invention, an improved attenuated strain of rabies virus having a significantly reproducible cytopathic activity is provided. The improved rabies virus strain used in this invention is prepared from the ERA strain of rabies virus. The ERA rabies strain has been identified by Abelseth, M. K. in “Propagation of Rabies virus in Pig Kidney Cell Culture”, Can. Vet. J. 584-87 (1964) and Abelseth, M. G., “An Attenuated Rabies Vaccine for Domestic Animals Produced in Tissue Culture”, Can. Vet. J. 5279-286 (1964) was derived from the rabies virus described by Fenje, P., in “Propagation of Rabies Virus in Cultures of Hamster Kidney Cells”. Can J. Microbiol, 6 379-484.
Reference may be made to Thoulouze et al., 1997, “Rabies virus infects mouse and human lymphocytes and induces apoptosis” in Journal of Virology 71: 10: 7372-7380, who showed that the rabies virus infects both mouse spleen lymphocytes and the human T-lymphocyte cell line. Jurkat found that attenuated rabies virus strain ERA infects ConA-activated splenocytes and T-cell lines more efficiently than CVS which is a highly neurovirulent rabies virus strain and reported that in contrast to CVS, ERA rabies virus and other attenuated viruses stimulate a strong immune response and can be efficient live vaccines. Both inactivated and attenuated viruses are used for immunization but the cost of production of inactivated virus is a major problem. Secondly, such a vaccine should be completely inactivated and for such purposes it is pre-requisite that candidate vaccine should be avirulent strain of the virus but it should be immunogenic and genetically stable. (Hooper et al., 1998, Collaboration of antibody and inflammation in the clearance of rabies virus from the CNS” in Journal of Virology; 72: 3711-9).
Yang et al., 1992 in Journal of General Virology; 73: 895-900, “Basis of neurovirulent rabies virus variant Av01 with sterotaxic brain inoculation in mice” reported that pathogenicity is not only a function of the virus but is also largely dependent on the site of infection and the immune status of the host. Even the most attenuated rabies viruses can potentially cause a lethal encephalomyelitis, which suggests that even inactivated and attenuated viruses are not reliable source for vaccination.
Reference may be made to Coslett et al., 1980, “The structural proteins of rabies virus and evidence for their synthesis from separate monocistronic RNA species” in Journal of General Virology, 49: 161-180 who reported that the genome of rabies virus is organized similar to that of vesicular stomatitis virus encoded by five major proteins, the nucleoprotein (N), phosphoprotein (NS) and polymerase (L) protein which, together with the genomic RNA form a nucleocapsid which is enveloped by a membrane (M) containing the transmembrane glycoprotein G. Conzehnann et al., 1990 in “Molecular cloning and complete nucleotide sequence of the attenuated rabies virus SAD B 19” Virology 175: 485-499 cloned and sequenced the complete nucleotide sequence of viral genome of SAD B 19 rabies virus strain which comprises 11, 928 nucleotides and encodes the five viral proteins N, NS M, G and L. The five cistrons are separated by intergenic regions of 2, 5, 5, and 24 nucleotides respectively.
Reference may be made to Gaudin et al, 1991 “Rabies Virus Glycoprotein is a Trimer” Virology 187, 627-632. They studied the oligomeric structure of glycoprotein both on the viral surface and after solubilization with detergents. The study shows that native quaternary structure of the glycoprotein is trimeric. However most detergents used in the study solubilized G in a monomeric form and only CHAPS a zwitterionic detergent, allowed solubilization of G in its native trimeric structure, this was determined using electron microscopy and sedimentation analysis of detergent solubilized G. The CHAPS solubilized G had a sedimentation coefficient of 9 S while other detergents solubilized G in a 4 S monomeric form. This study confirmed the results obtained by Whitt et al., 1991 “Membrane fusion activity, oligomerization and assembly of the rabies virus glycoprotein” in Virology 185, 681-688 wherein, a cross—linking reagent was used to study G expressed in HeLa cells from cloned cDNA. Electron microscopy also indicated that the native molecule has a “head” and a “stalk” and provided the basis for a low—resolution model of the glycoprotein structure.
Reference may be made to Dietzschold et al., 1983 “Characterization of an antigenic determinant of the glycoprotein that correlates with pathogenicity of rabies virus” Proc. Natl. Acad. Sci. USA; 80: 70-74 who reported that the amino acid arginine at 333 position of the glycoprotein plays a critical role in the pathogenicity of the rabies virus. Benmansour et al., 1991 “Antigenicity of rabies virus glycoprotein” Journal of Virology: 4198-4203 evaluated the relative importance of antigenic sites by describing more precisely the antigenicity of the protein by using monoclonal antibodies and neutralization—resistant (MAR) mutants. The 266 neutralizing monoclonal antibodies were identified in the study of which 97% belonged to sites II and III which were initially defined by Lafon et al., 1990 in “Human monoclonal antibodies specific for the rabies virus glycoprotein and N protein” Journal of General Virology; 71: 1689-1696.
Reference may be made from Cox et al., 1977 “Rabies virus glycoprotein 11 Biological and serological characterization” Infection Immunity 16, 743-759 wherein the glycoprotein of rabies virus was identified as a major antigen that induces protective immunity and induces the production of virus—neutralizing antibodies and confers immunity against a lethal challenge infection by the rabies virus. Although protection against rabies virus infection is probably the result of many host effector interactions, as studied by Turner 1985, “Immune response after rabies vaccination: basic aspects” Ann. Inst. Pasteur Virol. 136E: 453-460, the rabies virus G protein represents a logical choice for the development of a subunit vaccine that can be used for immunization against rabies in humans and animals. Reference in this respect can also be made from Cox et al., 1980, “Preparation and characterization of rabies hemagglutinin” Infection Immunity 30, 572-577.
Reference may be made to Swamy et al., 1984, “Neurological complication due to beta—propiolactone (BPL)—inactivated antirabies vaccination” Journal of the Neurological Sciences 63: 1: 111-128; who studied the neuroparalytic accident in patients due to antirabies vaccination with BPL vaccine which proves that the virus was not completely inactivated by beta-propiolactone and can be a high risk for the health because basically the inactivated rabies vaccines consist of suspensions of virus containing central nervous tissue of infected animals, such as rabbits or sheep or of suspensions of infected duck fetuses. Such type of vaccines also have a second drawback that, due to the high content of foreign proteins, they may cause undesired side effects at the point of injection as well as of general nature. When rabies vaccines from the central nervous tissue are used, the patient may even suffer neurocomplications with permanent damage, all the more since a series of injections is required for sufficient protection against rabies. Moreover, animal tissue based vaccines may carry other infective particles like prions and viruses like HIV, Fowl pox, Madcow etc.
Anilionis et al. 1981, “Structure of the glycoprotein gene in rabies virus” Nature: 294: 275-278 cloned the glycoprotein coding gene, using mRNA extracted from rabies virus infected BHK cells, purified by oligo (dT) cellulose chromatography and sucrose density centrifugation and determined complete nucleotide sequence of the glycoprotein cDNA. Subsequently, Yelverton et al., 1983, in “Rabies virus glycoprotein analogs: biosynthesis in Escherichia coli” Science: 219: 614-620 expressed the glycoprotein in Escherichia coli, but the protein was not immunologically active. This is because the antigenic determinants dependent on post translation modifications such as carbohydrate side—chain attachment, i.e. the introduction of the authentic carbohydrates, as it happens in eukaryotic cells is not achieved in Escherichia coli cells. Various other heterologous systems have been used to express the rabies glycoprotein. Two species of yeasts have been tried extensively for the expression of rabies glycoprotein. These are Saccharomyces cerevisiae (Kelper et al., 1993, Sakamoto et al., 1999) and Pichia pastoris (patent no. WO9000191, 1990). Polypeptides of 65-68 kDa, which migrated at the same molecular weight as authentic viral rabies G protein species, were synthesized by Saccharomyces yeast transformants as detected by immunoblotting with rabies specific antiserum. Reference may be made to Sakamoto et al., 1999, “Studies on the structures and antigenic properties of rabies virus glycoprotein analogues produced in yeast cells”, Vaccine: 17: 205-218 wherein two forms of rabies virus glycoprotein produced in the G-cDNA—transfected yeast cells were identified. These were designated as YGI (66 kDa) and YGII (56 kDa). The YG1 reacted with polyclonal anti-G antibodies but did not react with conformational epitope—specific MAb. While the protein expressed in Saccharomyces did not protect animals challenged with rabies virus, the protein expressed in Pichia were claimed to provide protection (WO 90000191 dated 1990). However, the details have not been published, nor has the Pichia system been used and may therefore not be a system of choice. The same G-cDNA was expressed in animal cells, a single form was produced. The results concluded that most G protein molecules were not processed normally in yeast cells. The crucial role of rabies glycoprotein in protection was determined by Foley et al., 2000 “A recombinant rabies virus expressing vesicular stomatitis virus glycoprotein fails to protect against rabies virus infection” in Proc. Natl. Acad. Sci.: 97; 26: 14680-14685, constructed a recombinant RV (rRV) in which rabies virus glycoprotein ectodomain and transmembrane domains were replaced with the corresponding regions of vesicular stomatitis virus (VSV) glycoprotein and immune response was studied and compared to parental rRV strain containing rabies virus glycoprotein. Similar immune responses against the internal viral proteins of both viruses indicated successful infection but all mice who received the rRV vaccine survived the challenge, whereas immunization with the domain substituted rRV-VSV-G did not induce protection. Which confirmed the critical and crucial role of glycoprotein of rabies virus and also demonstrated that immune response and immunoprotection against challenge with live rabies virus are two different phenomenon.
Expression of rabies glycoprotein gene by baculoviral vectors in insect cells gives high yields of protein to the extent of 18% of total cellular protein, 48 h post infection. In one study (Prehaud D H et al, 1989) the gene encoding the G protein of CVS strain was placed under the control of the AcNPV polyhedrin promoter and expressed at high levels by the derived recombinant virus using a Spodoptera fugiperda cell-line. The insect derived protein exhibited slightly faster electrophoretic mobility due to differences in the glycan components. Vaccination by insect derived glycoprotein, followed by challenged to mice gave delayed mortality, i.e., a low level of protection against rabies.
In another study, Rupprecht et al (1993) demonstrated that a glycoprotein (ERA strain) derived from recombinant baculovirus—infected insect cells was efficacious as an oral vaccine in raccoons. In view of relatively high costs of the insect and mammalian cell-systems these are not the systems of choice for G protein expression as a strategy to develop vaccine against rabies.
Some of the recombinant Pox virus rabies glycoprotein based vaccines licensed and marketed in US since 2000 are (i) Purevax for Feline rabies (cats)—monovalent, live canary pox vector (Merial Inc.) and (ii) Raboral V-RG for raccoons-oral live vaccinia vector. Recombinant Poxviruses (USPTO 5266313 dated 1986; USPTO 05348741 dated 1994) have many useful features as vectors for the expression of genes that carry immunizing antigens from other viruses. These are easy to produce and induce cellular and humoral immunity. However, there is concern about the safety of vaccinia virus, if used widely for men and animals.
A Canarypox virus Alvac-RG (vCP65) was used as a vector in non-avian species (Taylor et al., 1995) for expressing the rabies glycoprotein G gene (USPTO 5843456 dated 1998) to address the concern about the spread of vaccinia virus to non-target population, especially immunocompromised individuals. Avipox virus was also developed as a vector for expressing rabies glycoprotein gene. Avipoxvirus (canarypox) recombinants (USPTO 6340462 dated 2002) undergo abortive replication in nonavian cells, yet can achieve expression of extrinsic gene products and their presentation to the immune system. In vitro studies have shown that no replication of the virus can be detected on six human-derived cell lines, nor can the virus be readily adapted to replicate on non-avian cells. Expression of the rabies G was detected on all cell lines analyzed in the absence of productive viral replication. The safety and efficacy of the recombinant (Alvac-RG; vCP65) were tested in several animal species, then it was subjected to a phase 1 clinical trial. This study showed the potential of non-replicating poxviruses as vectors for vaccination in human beings (Fries et al., 1996). Recombinant were immunogenic by the intramuscular and subcutaneous routes. They were also immunogenic when given orally.
The viral vectors including posvirus vectors provide a convenient vehicle for the delivery of the vaccines. This also reduces the costs involved in purifying the proteins from cultures. However, owing to safety considerations, viral vectors meant for humans go through tougher scrutiny for approval by the regulatory bodies.
Cell-cultured based vaccines for rabies are limited to growing inactivated strains of the virus in cell cultures. These include, the relatively expensive Human Diploid Cell Vaccines (HDCV), purified Vero Cell Rabies vaccine (Verorab) and the more economical primary chick embryo cell culture vaccine (PCEV-Rabipur). These vaccines comprise the virus grown in cell cultures. Current biotechnological approaches aim at expressing the coat protein gene of the rabies virus to develop a safe RGP that could be deployed as an active vaccine.
The expression of proteins or safe ‘subunit vaccines’ in cell-cultures is a routine procedure in recent years. Examples of such useful host cell-lines are VERO and HeLa cells, Chinese Hamster Qvary (CHO) cell lines, and W 138, Baby Hamster Kidney (BHK), COS-7 and MDCK cell-lines. Stable expression of rabies virus glycoprotein was shown in Chinese Hamster Ovary cells (Burger et al., 1991). A full length, glycosylated protein of 67 K that co-migrated with the G-protein isolated from virus-infected cells, was obtained.
The rabies glycoprotein expressed in different cell-lines was similar to the native protein in terms of size and immunoreactivity. The recombinant protein thus produced provided an insight into the biology of the protein and gave a clue to the parameters to be taken into consideration while choosing an appropriate expression system. The proteins were of analytical grade and can be purified to a high degree of purity. However, the process based on animal cell lines is very expensive for industrial scale. In order to decide upon an economically viable alternative, the expression system should combine in itself the options for fermentor level scaling up (as in bacteria) and the option for producing a fully glycosylated protein that is closely similar to the native form (as in cell-lines). Expression in plants is a promising alternative in this respect.
Subunit vaccines are important improvements over conventional attenuated or killed vaccines in many aspects including safety and production systems. The expression of foreign proteins in plants has become an attractive alternative in recent years as it has the potential of producing recombinant proteins in large quantities and at low cost. Recent advances in genetic engineering have provided the requisite tools to transform plants to express foreign gene. Agrobacterium tumefaciens have proven to be efficient and highly versatile vehicles for the expression of industrially valuable foreign genes into the plant tissue, as described in Hood et al. (1999) “Plant—based production of xenogenic proteins” Current Opinion in Biotechnology 10: 382-386. Vaccine for human and animal disease prevention comprise the most competitive area for plant based production of xenogenic proteins. Utilization of plants as expression vectors for the production of foreign proteins has captured attention in recent years. Viral proteins (HbsAg, Norwalk virus capsid protein, rabies virus glycoprotein, FMDV structural protein VP1), bacterial toxins (LTB), antibody molecules and several other industrially and therapeutically important proteins have been expressed successfully in plants (Tacket et al., 2000; Kong et al., 2001). In most cases the expressed proteins are fully functional as antigens or in ligand recognition. Importantly, they are effective in eliciting in eliciting specific immune responses. The production of immunogens in plants might be an economic alternative to animal cell based production systems for the development of vaccine. The greatest advantage of using plant systems for the expression of therapeutically important proteins is the absence of human or animal pathogens like HIV, Fowl Fox, Mad Calf, prions etc. in the protein preparations made from plants. Yet another possibility is of utilizing the plant material directly as a feed, thus generating an edible vaccine. Although there is a lot of scope for studies on high—level protein accumulation, post—translational protein modifications and downstream processing, enough progress has been made to arouse interest in plants as robust and commercially viable systems.
The simplistic requirements of plants for sunlight, water and minerals makes them an inexpensive means of correctly processing and expressing proteins that can be quite complex. The traditional subunit vaccines are expensive to produce and not heat stable necessitating a ‘cold-chain’ en rote from manufacturer to vaccination. This limits their availability and use in low-funded health care systems of developing countries. However, proteins expressed in plant parts are often stable for years, as for example, the seed proteins.
A bacterial antigen (E. coli enterotoxin) produced in transgenic plants was shown to effectively immunize mice when the crude protein extracts from the transgenic plant tissue were administered orally, as shown by Curtiss and Cardineau, 1997 in “Oral immunization by transgenic plants” U.S. Pat. No. 5,686,079 and Haq et al., 1995 in “Oral immunization with a recombinant bacterial antigen produced in plants” Science 268: 714-716. The work of Haq and co-workers was followed by human clinical trials to show that humans do develop an immune response to antigen delivered in uncooked food as referred by Tackett et al., 1998 “Immunogenicity in humans of a recombinant bacterial antigen delivered in transgenic potato”. Nature Medicine 4: 607-609.
Reference may be made to McGarvey et al., 1995 “Expression of the rabies virus glycoprotein in transgenic tomatoes” Bio/technology 13: 1484-1487 who engineered tomato plants (Lycopersicon esculentum) to express a gene for the rabies glycoprotein (G-protein) under the control of the 35S′ promoter of cauliflower mosaic virus. The protein was expressed in tomato and showed molecular weight of 62 and 60 kDa in western blot after immunoprecipitation, as compared to 66 kDa observed for G protein from virus grown in BHK cells. The amount of G protein immunoprecipitated was found to be approximately 1-10 ng/mg of soluble protein i.e. at 0.0001% to 0.001% of soluble protein. The low expression level may have been due to using a poorly designed gene. For example, a native G protein coding gene was used along with its native signal peptide The inventors did not examine antigenicity of the G protein and therefore it is not possible to comment on biological activity and utility of the G protein expressed in tomato plant or the gene designed in that study, specially for therapeutic purpose.
Plant derived immune response against diseases such as mink enteritis and rabies were reported by expressing viral epitopes on the surface of plant viruses, followed by infection of susceptible host with the recombinant modified virus, reference can be made from Modelska et al., 1998 “Immunization against rabies with plant—derived antigen” Proc. Natl. Acad. Sci. USA 95: 2481-2485 and Yusibov et al., 2002, “Expression in plants and immunogenicity of plant virus—based experimental rabies vaccine”. The plant virus was purified from the tissue and administered to the test animals. Although this system is very effective, the size of the antigen polypeptide that can be expressed on surface of a vector virus is limited to 37 amino acids. Hence epitope mapping of the antigen is needed for this approach. Such thorough knowledge of the antigen is not generally available, especially with newly discovered diseases where the expression of full-length proteins may be the only option. Also several epitopes need to be identified and joined together since a single epitope may not give acceptable protection against challenge by the pathogenic virus. Furthermore, containment could be considered as a significant problem at the agricultural level, especially when environmentally stable viruses like TMV are used.